doi: 10.1152/ajpcell.00214.2005.
Epub 2005 Oct 19.
Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI
Affiliations
- PMID: 16236825
- PMCID: PMC1482466
- DOI: 10.1152/ajpcell.00214.2005
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Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI
Am J Physiol Cell Physiol.
2006 Mar.
Abstract
Signals generated by the extracellular matrix (ECM) promote cell survival. We have shown that chondrocytes detached from their native ECM and plated without serum at low density on poly-l-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-l-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-betaI. PKC-betaI was noted to translocate from the cytosol to the particulate membrane after plating on poly-l-lysine, and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-betaI activation and subsequent chondrocyte cell death. Cell survival on poly-l-lysine was significantly improved in the presence of oligomycin or DIDS, suggesting that ROS production occurred via complex V of the electron transport chain of the mitochondria and that ROS were released to the cytosol via voltage-dependent anion channels. Together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-betaI.
Figures
Reactive oxygen species (ROS) and chondrocyte survival on poly-l -lysine. A: cells were plated at low density in 24-well plates at 5 × 105 cells/ml (0.5 ml/well) in serum-free DMEM-Ham’s F-12 on poly-l -lysine-coated coverslips in the presence of various antioxidants for 24 h before cell survival was quantified using the LIVE/DEAD cell survival assay. Results represent means ± SE of 3 separate experiments using primary chondrocytes derived from different donors. *P < 0.001 vs. untreated control. MnTBAP, Mn(III) tetrakis(4-benzoic acid)porphyrin; NAC, N-acetyl-l -cysteine. B: cells were plated at low density in 96-well plates coated with either fibronectin or poly-l -lysine at 5 × 105 cells/ml (0.2 ml/well) in serum-free DMEM-Ham’s F-12 without phenol red. ROS were quantified with the ROS-sensitive dye 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) at a final concentration of 5 μM. Results represent means ± SE of 3 separate experiments using primary chondrocytes derived from different donors. *P < 0.001 vs. untreated control.
Effect of PKC inhibitors on chondrocyte survival on poly-l -lysine. Freshly isolated chondrocytes were plated on coverslips coated with either poly-l -lysine (A) or fibronectin (B) at 5 × 105 cells/ml (0.5 ml/well) for 24 h under serum-free conditions in the presence or absence of the PKC inhibitors Gö-6983, Ro 32–0432, and rotterlein. Survival was determined using the LIVE/DEAD cell survival assay. The IC50 of the inhibitors for each respective PKC isoform is shown at right. Results represent means ± SE of 3 separate experiments using primary chondrocytes derived from different donors. *P < 0.001 vs. untreated control.
PKC isoform translocation in chondrocytes plated on poly-l -lysine in the absence and presence of an antioxidant. Immunoblot analysis was used to detect the translocation of 10 PKC isoforms from the cytosol to the particulate membrane when freshly isolated chondrocytes were plated on poly-l -lysine in serum-free DMEM-Ham’s F-12 in the presence and absence of the antioxidant Tiron (125 μM). Cell lysates were prepared after culture for the indicated time points, and the particulate membrane was separated from the cytosol by ultracentrifugation. A: translocation of PKC-α and -βI. B: translocation of PKC-δ, -ε, -η, -θ, -ι, and -λ. No evidence of PKC-βII or PKC-γ was found in human articular chondrocytes by using immunoblot analysis (data not shown). Results are representative of at least 3 separate experiments that gave similar results.
Quantitation of apoptotic and necrotic death of chondrocytes plated on poly-l -lysine. Freshly isolated chondrocytes were plated on poly-l -lysine or fibronectin as described for Fig. 1. At the indicated time points, the cells were sequentially stained with Trypan blue, fixed with 4% paraformaldehyde, and, finally, TdT-mediated dUTP nick end labeling (TUNEL) stained as described in materials and methods . Cells that were both Trypan blue and TUNEL positive were considered to be apoptotic, whereas cells that were Trypan blue positive and TUNEL negative were considered to be necrotic. At least 100 cells were counted for each time point, and the results are reported as the percentage that showed positive staining. A: cells staining with Trypan blue after plating on either poly-l -lysine or fibronectin. B: cells quantified with both Trypan blue and TUNEL staining after plating on poly-l -lysine. C: cells were plated on poly-l -lysine with or without 125 μM Tiron (to promote survival) or on fibronectin with or without 100 nM phenylarsine oxide (PAO; to induce apoptosis). DNA fragmentation was measured at the indicated time points using an ELISA for histone-associated DNA fragments. OD, optical density.
Chondrocyte death on poly-l -lysine does not require caspase activity. Freshly isolated chondrocytes were plated on either poly-l -lysine (A) or fibronectin (B) for 24 h under serum-free conditions, as described in Fig. 1, in the absence or presence of a broad caspase inhibitor (VAD) and/or a caspase-3-specific inhibitor (DEVD). Survival was determined using the LIVE/DEAD cell survival assay. Data presented in this section represent typical results from the dose-response experiments. Cells also were plated on poly-l -lysine with or without Tiron (to promote survival) or on fibronectin with or without PAO (to induce apoptosis), and caspase activity was measured using an assay for general caspase activity (C) or caspase-3 activity (D). Results represent means ± SE of 3 separate experiments.
ATP levels in chondrocytes plated on poly-l -lysine or fibronectin. Freshly isolated chondrocytes were plated on either poly-l -lysine- or fibronectin-coated 12-well plates, and at the indicated time points, ATP levels were measured using a luciferase-based assay. Results represent means ± SE of 3 separate experiments. *P < 0.001 vs. fibronectin.
Theoretical model for pathways resulting in chondrocyte death when matrix and growth factor survival signals are lacking in cells plated under serum-free conditions on poly-l -lysine. Mito, mitochondria.
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